Mononuclear phagocytes (monocytes) in their various forms have been shown to participate in many critical phases of the mammalian immune response. Monocytes and macrophages are known to be essential for the initiation of immune responses by virtue of their ability to process antigen (Rosenthal, New Engl. J. Med. 303, 1153, 1980), and for their ability to secrete soluble factors such as interleukin 1 (IL-1), colony stimulating factor (CSF), interferon (IFN) and prostaglandin E (PGE) which allow them to function as immunoregulators for a number of immune responses (Epstein, Biology of Lymphokines; Academic Press, NY, pp. 123-152. 1979, Stevenson, The Reticuloendothelial System. A Comprehensive Treatise, Vol. VI: Plenum Press, NY, pp. 79-91. 1982). In addition, monocytes are known to play a critical role as final effector cells in humoral immunity by virtue of the fact that these cells secrete complement components (Nathan, et al, New England J. Med. 303, 623. 1980) and are capable of mediating cytotoxic functions. In addition to antibody-dependent cellular cytotoxicity (ADCC) (Poplack, et al, Blood 48, 890. 1976), activated killer monocytes (AKM) are known to be potent killers of tumor cells (Stevenson, et al, Artificial Organs 112, 128. 1988).
Assessment of the in vitro function of human monocytes and AKM has been hampered by a number of technical and theoretical problems. First, monocytes constitute a very low proportion of the cells in human peripheral blood (generally less than 5%); thus, obtaining large numbers of them has been quite difficult. In addition, very few techniques have emerged which allow for the large-scale isolation of purified populations of human monocytes by negative selection; instead, generally small numbers of rather impure monocytes are isolated on gradients such as Percoll (Hester, et al., 1981) or cells of higher purity are obtained by adhering them onto plastic or glass labware by positive selection (Werb, J. Exp. Med. 147, 1695. 1978).
When monocytes are obtained by positive selection, it is difficult to remove them for further study; a variety of rather harsh measures are utilized to remove the adhered cells from the plastic or glass surface, ranging from the use of rubber policemen (Pennline. Manual of Macrophage Methodology, pp. 65-77. 1981), EDTA (Ackerman and Douglas, J. Immunol. 120, 1372. 1979), and lidocaine-containing solutions (Koski, et al, In Vitro Methods in Cell-Mediated and Tumor Immunity, Academic Press, p359. 1976). The potential problems of adherence and positive selection are compounded when the cells are activated in culture into AKM since most researchers employ some sort of polystyrene labware to which the monocytes will readily adhere.
Adherent monocytes, of course, are in a different condition from their normal state of suspension in human peripheral blood. Therefore, the functions may also be different. Furthermore, when placed in medium conditions under which they are generally cultured, human monocytes demonstrate a number of technical inadequacies. These problems mainly stem from the limited nutrients provided in most standard laboratory culture media for a cell as metabolically active as the human monocyte, and the additional potential artifacts created by culturing human monocytes with sera from different human individuals (AB serum) or from other species (such as fetal calf serum). Thus consistent and uniform conditions for culturing human monocytes and transforming them into AKM cannot be assured from batch to batch.
The above-cited problems regarding the handling of human monocytes are compounded when these cells are transformed into activated killer monocytes (AKM) for the treatment of cancer. Prior to the present invention, it has not been possible to prepare AKM in suspension suitable for administration to cancer patients for therapy. Moreover, no method for monitoring the cytotoxic activity of serum-free, suspension cultured AKM in vitro existed.